translational repression and eif4a2 activity are critical for microrna-mediated gene...
DESCRIPTION
Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-Mediated Gene Regulation. This assessed presentation looked at the effects of miRNA-mediated repression on mRNA. These effects include deadenylation, translational inhibition and degradation. It was observed that the eIF4A2 is a required component in the repression pathway mediated by the miRNA let-7. Presentation by KATE, Wisdom Deebeke; Mene Nkue; and Michael Thirlwell. Dated: Thursday, December 5th, 2013. (Phase 2, stage 3, semester 1).TRANSCRIPT
BGM3055 Presentation on
Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-
Mediated Gene Regulation
Presenters:Michael Thirlwell, KATE Wisdom Deebeke, and Mene Nkue.
Aims and Objectives:• To discuss the General concept of Translational
repression• To describe the methods used in this study• To analyse the results and evaluate the outcome of the
experiments
RNAi Timeline 1990: Introduction of gene copies into Petunia led to patchy plants.
1993: First miRNA discovered in C. elegans
1998: Injection of dsRNAs into C. elegans led to silencing of complementary genes
2000: Second miRNA discovered in C. elegans
2004: RNAi review paper claims: “Arguably the most important advance in biology in decades has been the discovery that RNA molecules can regulate the expression of genes.”
Novina et. al 1990
siRNA dsRNA can be produced by exogenous sources (e.g. virus) orfrom a transgene or a rogue genetic element.
dsRNA is cleaved into fragments (20-25 bp) by Dicer
RISC binds fragments and degrades sense strand
Remaining antisense strand hybridizes with mRNA
RISC degrades mRNA
miRNA Genome codes for transcription of pri-miRNAs which due to imperfect self complementarity fold into a hairpin
Drosha cleaves ends to form pre-miRNAs
Dicer cleaves into 21-22 bp single stranded miRNAs
miRNA forms miRNP which then binds mRNA imperfectly, and so inhibiting translation
Rarely hybridises perfectly and soleads to mRNA degradation
Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-Mediated Gene Regulation
This paper studied the mechanism of miRNA(let-7) inhibition of mRNA
Found that unadenylated and polyadenylated mRNAs are repressed
Greater magnitude of repression when mRNA is polyadenylated
mRNA Destabilization follows Repression.
Discovered that “eIF4A2 plays a critical role early in the repression pathway”
Methods
Plasmid constructs:• Designed different plasmid constructs (pRL, pRL-Let-7
and pRL-Let-2) for different experiments:
• IRES for pRL-HCV/let-7, pRL-EMCV/let-7 and pRL-CrPV/let-7 • ß-globin 5’UTR or the (CAA)18
5’UTR• SiRNA generated against specific
translation initiation factor proteins• Used specific RE to digest plasmids
and insert the ‘’insert’’
Fig. Taken from Promega website (promega.co.uk)
Methods continued
In vitro transcription and polyadenylation
(with the exception of globin-pRL/Let-7 and CAA-pRL/Let-7)
– Linearised plasmid with BamHI followed by Agarose gel purification.
– Polyadenylation with poly(A) tailing kit
Fig. Taken from 5PRIME website (www.5prime.com)
Methods continued
• Different mRNAs transcribed:
Meijer et al., Science 340:82-5 (2013)
Schematic representation of RNA used for transfection.
RNA used in deadenylation study
IRES-containing RNA used for the transfection study
Schematic representation of RNA transcribed from globin and CAA18 plasmids
Methods continued Transfected plasmids intoHeLa, HEK293 or HCT cells
byLipofectamine2000, fugeneor Genejammer methods
Other assays include:• Luciferase assay (assayed for
Rluc relative to Fluc)• Immunoprecipitation (WB for
transfected eIF4A1/2)• RNA isolation, RT-qPCR and
PAT assays
Fig. Taken from: http://www.bocascientific.com
Translational Repression contributes to miRNA mediated gene regulation
Repression of translation by let-7 miRNA increases with time. CrPV: no initiation factor HCV: requires only eIF(2,3,5) eIF5b & Met tRNAi
EMCV: requires all initiation factor except 4E Translational repression is required for miRNA-mediated control elF4F is essential for repression mRNA destabilisation follows translational repression
Meijer et al., Science 340:82-5 (2013)
eIF4F is required for miRNA mediated mRNA repression
Beta globin 5’UTR unstructured(CAA)18 5’UTR (does not require unwinding) unstructured 5’UTR was refractory to miRNA-mediated repression, showed slight activation
showed that 4A is the implicated EIf4f required for miRNA-mediated translational repression
EIf4A2 is the only component of elF4F required for miRNA-mediated repression
siRNA was used to knockdown the expression of eIF4F components
after depletion of eIF4A2, only eIF4A2 and not eIF4A1 can restore repression
eIF4A2 is the key factor for miRNA-mediated gene silencing.
Conclusion Brief history of RNAi (miRNA and siRNA), biological
application
Repression of translation by Let-7 miRNA
Key methods used to achieve repression of mRNA translation by the miRNA Let-7
Requirement of translational repression and the eIF4A2 for miRNA-mediated gene control
References
• Meijer, H.A., Kong, Y.W., Lu, W.T., Wilczynska, A., Spriggs, R.V., Robinson, S.W., Godfrey, J.D., Willis, A.E. and Bushell, M. (2013) 'Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-Mediated Gene Regulation', Science, 340(6128), pp. 82-85.
• Fabian, M.R., Sonenberg, N. and Filipowicz, W. (2010) 'Regulation of mRNA Translation and Stability by microRNAs', Annual Review of Biochemistry, 79(1), pp. 351-379.
• Fabian, M.R., Mathonnet, G., Sundermeier, T., Mathys, H., Zipprich, J.T., Svitkin, Y.V., Rivas, F., Jinek, M., Wohlschlegel, J., Doudna, J.A., Chen, C.-Y.A., Shyu, A.-B., Yates, J.R., Hannon, G.J., Filipowicz, W., Duchaine, T.F. and Sonenberg, N. (2009) 'Mammalian miRNA RISC Recruits CAF1 and PABP to Affect PABP-Dependent Deadenylation', Molecular cell, 35(6), pp. 868-880.